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Modification of thread-based microfluidic device with polysiloxanes for the development of a sensitive and selective immunoassay
Accepted Manuscript Title: Modification of thread-based microfluidic device with polysiloxanes for the development of a sensitive and selective immunoassay Authors: Jane Ru Choi, Azadeh Nilghaz, Lei Chen, Keng C. Chou, Xiaonan Lu PII: DOI: Reference:
S0925-4005(18)30103-5 https://doi.org/10.1016/j.snb.2018.01.102 SNB 23951
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
7-9-2017 17-12-2017 9-1-2018
Please cite this article as: Jane Ru Choi, Azadeh Nilghaz, Lei Chen, Keng C.Chou, Xiaonan Lu, Modification of thread-based microfluidic device with polysiloxanes for the development of a sensitive and selective immunoassay, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.01.102 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Modification of thread-based microfluidic device with polysiloxanes for the development of a sensitive and selective immunoassay
Jane Ru Choi 1, Azadeh Nilghaz 1, Lei Chen 2, Keng C. Chou 2, and Xiaonan Lu 1*
Food, Nutrition and Health Program, Faculty of Land and Food Systems, The University of British
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Columbia, Vancouver, BC, V6T 1Z4, Canada
Department of Chemistry, The University of British Columbia, Vancouver, BC, V6T 1Z1, Canada
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Corresponding author. Email address: [email protected]
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Research highlights
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Graphical abstract
Polysiloxanes can be tuned to manipulate the fluid flow in thread-based microfluidic devices
Polysiloxanes-modified device enables optimum interaction between antigen and antibody
This device can sensitively detect bacteria in diverse real samples
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This work expands the application of polysiloxanes in microfluidics
ABSTRACT
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We demonstrate that polysiloxanes can be tuned to be partially hydrophilic for manipulating the fluidic flow in the pores of cotton thread-based microfluidic devices. A mixture of methanol and isopropanol was used as a diluent for siloxane precursor, which was included into the thread to enable rapid curing of polysiloxanes for fluidic control and enhance detection sensitivity. Interestingly, twelve-fold diluted polysiloxanes enabled desirable fluidic delay and optimum interaction between the targeted antigen
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and detection antibody-gold nanoparticles (dAb-AuNPs) in the thread-based immunoassay, generating more antigen-dAb-AuNP complexes that bound to the capture antibody (cAb) at the test zone and
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achieved signal enhancement (~10-fold over unmodified device). The phenomenon of fluidic delay was evaluated by mathematical simulation, through which the fluidic movement on the polysiloxanescoated region was observed and the simulation data was in agreement with the experimental data. This
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polysiloxanes-modified device could detect Salmonella enterica serotype Enteritidis (as a model
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analyte) in phosphate buffered saline, spiked whole milk, juice and lettuce with the detection limit of
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500, 1000, 1000 and 5000 CFU/mL, respectively, which was comparable to or even more sensitive
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than the existing immunoassays. This work expands the application of polysiloxanes in the
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microfluidic devices for biomedical diagnosis, water quality monitoring, and food safety surveillance.
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immunoassay
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Keywords: polysiloxanes, thread-based microfluidic devices, fluidic control, sensitivity enhancement,
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1. Introduction With the advances in point-of-care (POC) technologies, paper-based and textile-based microfluidic devices have become attractive platforms for nucleic acid testing and immunoassays for a wide range of applications (e.g., biomedical diagnosis, food safety surveillance, and environmental monitoring) [1]. In general, these POC assays are complementary to the conventional labor-intensive, time-
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consuming and expensive laboratory diagnostic assays, such as enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) technique [2, 3]. Textile-based
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devices (e.g., thread-based devices) are of extremely lower-cost (<$1 USD per test) and require a lower sample volume (~20 μL) for testing compared to lateral flow test strip (require ~50 μL sample, $3 USD per test) [4]. The nitrocellulose membrane of lateral flow test strip made up of cellulose nitrate
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is more fragile and inherently brittle under tension compared to thread. Similar to paper substrates, the
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cotton thread has excellent capabilities of immobilizing biomolecules (e.g., proteins and nucleic acids)
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and transporting fluid via capillary action, hence it is highly suitable for rapid detection and analysis
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of various analytes [5]. The excellent properties of thread make it a promising alternative to the
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conventional lateral flow test strips [5-7]. However, similar to paper-based microfluidic devices, the relatively poor sensitivity of the thread-based microfluidic devices has limited their wide applications
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[8].
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Polysiloxanes are inorganic polymers that consist of repeating units of siloxane and also known as silicones. They have the ability to resist to breaching by liquids and surfactants with low surface energy
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[9]. Their compatibility to various chemicals makes them a possible candidate to be included into the microfluidic devices for the development of various chemical and biological assays. Other hydrophobic materials, such as wax and alkyl ketene dimer, are commonly used in the development of different microfluidic assays [10]. However, they are unable to resist to breaching by the low surface energy surfactants [9]. The examples of surfactants include anionic (e.g., sodium dodecyl sulfate),
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non-ionic (e.g., Triton X-100) and cationic (e.g., cetyltrimethylammonium bromide) surfactants, which are commonly used as lysis reagents in most biological assays [11, 12]. In contrast, polysiloxanes has been reported to have the ability to resist to breaching by these surfactants due to its relatively lower surface energy [9]. For the synthesis of polysiloxanes, the Piers-Rubinsztajn reaction utilizes a Lewis acidic boron catalyst to facilitate the condensation of hydrosilanes with alkoxysilanes
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that can form new siloxane bonds [13]. This reaction eventually leads to the production of polysiloxanes only in a few seconds or minutes at room temperature [9]. Despite its ability to create
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hydrophobic zones and channels in the microfluidic assays due to the aforementioned properties, the possibility of tuning its hydrophobicity in the microfluidic assays has remained unexplored yet. Considering the potential of tuning its hydrophobicity, incorporating it into the thread-based device
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can potentially achieve desirable fluidic control and optimum device performance at a minimal cost.
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In the current study, we demonstrated a new approach of tuning the hydrophobicity of polysiloxanes
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to manipulate liquid flow in a thread-based microfluidic assay. A mixture of methanol and isopropanol
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was used as a diluent for siloxane precursor, which was then incorporated into the cotton thread-based devices to enable rapid curing of polysiloxanes for fluidic control within their pores. Interestingly, we identified that the delay of fluid flow was dependent upon the concentration of polysiloxanes, and the
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controlled flow increased the interaction between the targeted antigens and detection antibodies-gold
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nanoparticles (dAb-AuNPs) in thread-based assays, forming more antigen-dAb-AuNP complexes. These complexes eventually bound to the capture Ab (cAb) at the test zone and enabled ~10-fold signal
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enhancement over the unmodified thread-based immunoassay. The phenomenon of fluidic delay was evaluated by mathematical simulation through which the fluidic movement on the polysiloxanescoated region was observed and the simulation result was in agreement with the experimental data. As food microbiological safety remains a major concern of public health in both developing and developed countries, the application of our developed device for food safety analysis was evaluated
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by detecting foodborne pathogens in spiked drink and food samples. Salmonella enterica serotype Enteritidis was used as a model bacterium in this study given that it is one of the most common causes of foodborne illnesses worldwide [14]. S. Enteritidis could be detected by using this polysiloxanesmodified thread-based device in phosphate buffered saline, spiked whole milk, juice and lettuce with the detection limit of 500, 1000, 1000 and 5000 CFU/mL, respectively, which was comparable to or
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even more sensitive than the current POC immunoassays. The good specificity of this proposed immunoassay was evidenced by only positive result shown in Salmonella-positive samples. In
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comparison, other bacterial samples (i.e., Escherichia coli, Campylobacter, and Listeria monocytogenes) and controls [i.e., distilled water and phosphate buffered saline (PBS)] showed
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negative results.
2. Materials and Methods
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2.1. Bacterial strains and cultivation. S. Enteritidis ATCC 43353, E. coli O157:H7 ATCC 43894, C.
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jejuni F38011 (a clinical isolate) and L. monocytogenes ATCC 19113 were used in the current study.
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S. Enteritidis and E. coli O157:H7 were cultivated in tryptic soy broth (BD Difco) while L. monocytogenes was cultivated in Brain Heart Infusion (BHI) broth (BD Difco) in an aerobic condition. C. jejuni was cultivated in Mueller Hinton broth (BD Difco) in a microaerobic condition (85% N2,
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10% CO2, 5% O2). The cultivation temperature of all the bacteria was set at 37°C.
2.2. Preparation of gold nanoparticles and gold nanoparticles-antibody conjugates. Gold
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nanoparticles (AuNPs) with an average diameter of 13±3 nm were prepared according to the protocol described in previous publications [1, 2]. Briefly, 4.5 mL of 1% trisodium citrate solution and 1% chloroauric acid (HAuCl4) was rapidly added into a round bottom flask with 100 mL of boiled distilled water. The color of the solution immediately turned from yellow to purple and finally to wine red in a few minutes.
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2.3. Preparation of gold nanoparticles-antibody conjugates. The optimum pH value of 8 was achieved by adding 4 µL of potassium carbonate into the AuNP solution. Different volumes of dAb (anti-S. Enteritidis mouse monoclonal Ab, 0, 1, 2, 3, 4, and 5 µL) (VWR Scientific Canada) were added into the AuNP solution in different tubes. Then, 10% (w/v) BSA was added to block non-
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specific binding sites. The supernatant was removed after the centrifugation at 12,000 ×g for 10 min, and the red pellets were suspended in 0.22 mL of eluent buffer that consists of 1% BSA, 0.85% Tris,
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5% trehalose, and 20% sucrose. The AuNPs were characterized using transmission electron microscopy (TEM) whereas the AuNP-antibody conjugates were characterized by the visible colour changes from wine red to dark red and a slight shift of the absorbance values in ultraviolet-visible
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spectrophotometry.
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2.4. Fabrication of the thread-based microfluidic device. The thread-based microfluidic device was
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fabricated according to the protocols described in a previous publication with modifications [3].
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Briefly, the thread-based device consisted of three major components: a sample pad, a cotton thread and an absorbent pad. Cotton threads were treated to be hydrophilic with air plasma (PDC-32G, Harrick Plasma, Ithaca, NY). A test zone and a control zone were defined on each thread. The test
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zone was coated with 0.6 µL (applied as three aliquots of 0.2 µL, with 10 min drying at 37°C after the
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addition of each aliquot) of cAb against S. Enteritidis at a concentration of 1 mg/mL in PBS containing 15% glycerol. The control zone was coated with anti-mouse IgG (0.6 µL at 1 mg/mL) (VWR Scientific
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Canada). The antibody was applied as three aliquots of 0.2 µL, with 10 min drying at 37°C after the addition of each aliquot. Five microliters of AuNP-Ab were added onto the sample pad and dried at 37°C for 1 hr before use. Double-sided tapes were placed on the backing pad in parallel ways with a distance up to 4 cm. The thread was then mounted onto the double-sided tapes. A piece of glass fiber
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that served as sample pad (10 × 10 mm) was placed at one end of the thread whereas a piece of cellulose pad (10 × 20 mm) that served as absorbent pad was placed at the other end.
2.5. Preparation of the polysiloxane-modified thread-based microfluidic device. Polysiloxane was used to control the fluidic flow of the immunoassay. This hydrophobic agent was prepared based upon
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the protocol described in a previous publication with modifications [3]. Briefly, 0.004 mmol tris(pentafluorophenyl)borane [B(C6F5)3] was added to 1,3-dimethyltetramethoxysilane (DMTMDS)
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(0.6 mmol) to form the stock solution. The stock solution was mixed with the appropriate amount of methanol-isopropanol mixture and finally with tetrakis(dimethylsiloxy)silane (QMH4, Gelest). Different concentrations of hydrophobic mixture (0.5 µL) were then individually dispensed into the
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threads.
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2.6. Optimization of the thread-based immunoassay. In the optimization assays, the structure of
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thread-based device, the volume of dAb-AuNP, as well as the concentration of glycerol and cAb at the
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test zone were optimized to achieve the optimum colorimetric signal at the test zone. Different constructions of the thread-based device were performed, especially on both test zone and control zone in order to minimize the spreading area of both cAb and control Ab and simplify assay readout. To
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optimize the concentration of dAb-AuNP, different volumes of dAb-AuNP (1, 2, 3, 4, and 5 µL) were
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individually tested and the optimum volume of dAb-AuNP was determined. To optimize the concentration of glycerol at the test zone, different concentrations of glycerol (5%, 10%, 15%, and
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20%) were individually mixed with cAb and the corresponding color intensity was observed. Different concentrations of cAb at the test zone (0.5, 1, 1.5, and 2 mg/mL) were also individually tested in the immunoassay. The sensitivity of the assay was determined based upon the color intensity at the test zone and the optimum parameters were determined.
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2.7. Thread-based lateral flow immunoassay for the detection of S. Enteritidis. To perform the lateral flow immunoassay, 20 µL of sample was applied onto the sample pad. Sensitivity assay was performed with different concentrations of polysiloxanes using a mixture of methanol and isopropanol as the diluent. The specificity of the assay was determined by testing the device using different species of bacteria (i.e., E. coli O157:H7, L. monocytogenes and C. jejuni). The color changes at both test zone
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and control zone were observed in 10 min. For quantitative analysis, images of the test zones were
ProPlus 6.0 software (Media cybernetics. Inc., Bethesda, MD).
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captured using a smartphone (IPhone 6S) and their optical densities were determined using the image
2.8. Mathematical simulation. To mathematically simulate the fluidic delay due to polysiloxanes in
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the thread, a physical model for a steady-state flow was developed in the current study. Brinkman
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equation was used to describe the fluidic flow in this porous material and the viscosity effect is taken
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into consideration:
, where
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(1)
, p, µ, µe and K are fluid velocity, pressure, fluid viscosity, effective viscosity for Brinkman
term and permeability of porous medium, respectively. The fluid viscosity (µ) corresponds to that of
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PBS (as a diluent) at room temperature. The effective viscosity (µe) corresponds to the fluid viscosity
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(µ) in the porous medium [15]. The permeability K is given as follows [16]:
K =r 2
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1.5
2
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, where ε is the porosity of the material and r is the average fiber radius. The porosity ε was obtained by measuring the volume of the liquid absorbed by the materials based upon the liquid-displacement method with modifications [17]. The average fiber radius r for each group were obtained from the 9
scanning electron microscopy (SEM) images, which were 17±0.12 µm, 13.3±0.15 µm, 12±0.26 µm, 11.23±0.15 µm, 10.4±0.12 µm and 8.2±0.34 µm for unmodified thread and the threads incorporated with 8-fold, 10-fold, 12-fold, 14-fold and 16-fold dilutions of polysiloxanes, respectively. As for the boundary conditions, the inlet velocity was calculated with the known sample volume (20 µL), the inlet cross-section and the fluid absorption period. The outlet pressure is equivalent to the atmospheric
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pressure. All other bounding walls are under non-slip conditions. The mathematical simulation was
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performed using the Brinkman equation module of Comsol Multiphysics 5.0 software.
2.9. Detection of S. Enteritidis in different types of food samples. After overnight cultivation, bacteria were first diluted in PBS for optimization of the thread-based assays. To further investigate
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the ability of the device to detect pathogens in agri-food products, we spiked S. Enteritidis into milk,
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juice and lettuce obtained from a local grocery store in Vancouver, Canada. Each sample was first
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confirmed to be without contamination of Salmonella, followed by spiking different concentrations of
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S. Enteritidis (i.e., ranging from 1 to 105 CFU/mL). Lettuces were washed and then spiked with S.
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Enteritidis before mixing with 100 mL of ultrapure water in a blender. The mixture was then filtered through a 100-µm filter paper to remove the debris prior to the detection by the immunoassay.
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3. Results and Discussion
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3.1. Working principle of polysiloxanes-modified thread-based microfluidic device. Taking the advantage of polysiloxanes that resist to the surfactants, we propose that its hydrophobicity can be
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tuned and its incorporation into the thread-based microfluidic device can manipulate fluid flow and subsequently achieve the optimum performance of the device. The Piers-Rubinsztajn reaction was carried out for the formation of silicone elastomer to prepare polysiloxanes [13]. This reaction is rapid and utilizes a Lewis acidic boron catalyst namely tris(pentafluorophenyl)borane, B(C6F5)3 to facilitate the condensation of hydrosilanes [tetrakis(dimethylsiloxy)silane] with alkoxysilanes (1,3-
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dimethyltetramethoxysilane) to form new siloxane bonds (R3SiH + R3SiOR’ → R3SiOSiR3 + R’H) [9, 18]. As aforementioned, a mixture of methanol and isopropanol (1:1) was used as a diluent for siloxane precursor as it can ideally control both viscosity and surface tension of the final products, namely polysiloxanes. By tuning the amount of diluent, we propose that the hydrophobicity of polysiloxanes can be tuned to enable a well-controlled fluidic flow in the thread-based microfluidic
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device.
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Similar to paper-based immunoassays, thread-based immunoassays consist of two major zones, namely test zone and control zone. The cAb and control Ab were dispensed onto the test zone and control zone, respectively. Cotton thread used in the study is mainly made up of cellulose. There are
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high density of hydroxyl groups on the surface of cellulose that allow the irreversible binding of
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capturing antibody to the surface of cotton thread through a combination of electrostatic and
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hydrophobic interactions [19, 20]. These interactions are sufficiently strong to resist to the fluidic
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movement. The AuNPs used in this study were characterized using transmission electron microscopy
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(TEM) (Figure S1A). The AuNP-antibody conjugates were characterized by the visible colour changes from wine red to dark red (Figure S1B) and a slight shift of the absorbance values (7 nm) in
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ultraviolet-visible spectrophotometry (Figure S1C). The particle size was 13 nm (Figure S1D).
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The cotton thread is attached to the sample pad that allows sample addition and AuNP release as well as absorbent pad that facilitates fluid flow. Polysiloxanes was added onto the region between sample
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pad and test zone to create fluidic delay. Twenty microliters of sample (i.e., Salmonella) were applied onto the sample pad where dAb-AuNP was dried. The antigen-dAb-AuNP complexes then flowed across the cotton thread and bound to the cAb on the test zone to form colorimetric band. The color intensity of the test zone was directly proportional to the concentration of the analytes (i.e., Salmonella). The excessive dAb-AuNP was bound to the control zone and formed another colorimetric
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band (Figure 1).
3.2. Optimization of the thread-based immunoassay. To reduce the spreading area of cAb and control Ab at both zones, the regions of the test zone and control zone were modified. Both zones should be small and concentrated to minimize antibody spreading in the immunoassay. Unlike
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previous publications that demonstrated the direct dispension of antibodies onto the thread [19, 21, 22], we modified both zones by creating knots to reduce the spreading area of antibodies (Figure S2).
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To create a knot, one end of the thread was inserted into the loop and both ends were pulled together in the opposite direction to tighten it. The other knot was also created using the same method. This modified structure simplifies the readout and assay analysis by clearly defining the region of the
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colorimetric signal. We also dispensed three aliquots (0.2 µL each) to further minimize the wicking of
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the antibody solution along the thread. To determine the amount of dAb-AuNP that was required to
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achieve the optimum colorimetric signal at the test zone, different volumes of dAb-AuNP (i.e., 1-5
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µL) were individually tested (Figure S3). The amount that required obtaining an optimum signal in
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the immunoassay was determined to be 4 µL. A smaller volume of the dAb-AuNP produced a weaker signal at the test zone while a larger volume yielded an increase in the background signal.
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To determine the concentration of the glycerol that acts as a diluent of dAb-AuNP, we optimized its
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concentration in the range of 5% - 20%. In general, 15% of glycerol produced an optimum signal at the test zone. Glycerol with a lower concentration (<15%) yielded a weaker signal while glycerol with
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a higher concentration (i.e., 20%) resulted in an increase in the background signal. This might be due to the significantly increased viscosity at the test zone by the treatment of a high concentration of glycerol, leading to the failure of considerable amount of antigen-dAb-AuNP complexes to completely wick through the thread that subsequently generated a false positive result (Figure S3).
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The concentration of the immobilized cAb at the test zone was also tested in the range of 0.5-2 mg/mL (Figure S3). Along with the increased concentration of cAb, the color intensity of the test zone increased. However, a concentration over 1.5 mg/mL (i.e., 2 mg/mL) produced a false positive result with a higher background signal, which might be due to the significant amount of cAb that reduced the flow rate of antigen-dAb-AuNP complexes. Therefore, 1.5 mg/mL was selected as the optimum
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concentration of cAb. The optimized conditions (i.e., 4 μL dAb-AuNP, 15% glycerol and 1.5 mg/mL immobilized cAb) with the modified “knot” structure of the thread were then used for the subsequent
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assays, which were able to produce intensive color at the test zone with negligible background signal.
3.3. Integrating polysiloxanes into thread-based microfluidic device to create fluidic delay. We
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utilized the optimum volume of polysiloxanes (0.5 μL) to completely cover the biomolecule interaction
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region (i.e., the entire region prior to the test zone) for desirable manipulation of the flow rate (Figure
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S4). To evaluate the effect of polysiloxanes on thread-based immunoassay, polysiloxanes were
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dispensed into the thread prior to running the assay. To determine the range of the concentration
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required to allow the fluid to wick through the entire thread, we performed the immunoassay with polysiloxanes in the concentration range of 4-fold to 16-fold dilution (Figure 2A). The concentration of polysiloxanes with at least 8-fold dilution allowed the solution to completely flow through the
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thread. The 4-fold diluted polysiloxanes was too concentrated to allow the complete flow of liquid
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within the thread. Therefore, we utilized polysiloxanes with the concentration range of 8-fold to 16-
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fold for the development of this immunoassay.
To study the effect of different concentrations of polysiloxanes on the hydrophobicity of the thread, contact angle measurements were performed. In the absence of polysiloxanes (negative control), water droplets were identified to immediately wick into the thread. In contrast, the highly concentrated polysiloxanes allowed the thread to be more hydrophobic, thereby a water droplet being formed on the
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surface of the thread with an apparent contact angle. The contact angle of droplets was 89.6 ± 6°, 65.7 ± 2°, 56.3 ± 1°, 52.2 ± 6° 46.5 ± 4° and 34.9 ± 8° for 4-fold, 8-fold, 10-fold, 12-fold, 14-fold and 16fold dilution of polysiloxanes, respectively, at 2 s after the addition of 5 μL of liquids (Figure 2B). Taken together, the lower the concentration of polysiloxanes on the thread, the lower the water contact angle, indicating less hydrophobicity of the polysiloxanes-coated thread. To study the hydrophobicity
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of polysiloxanes on the flow rate, different dilutions of polysiloxanes were applied onto the thread and their flow distance over time was correspondingly recorded. Based upon the Washburn’s equation
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[23], the flow distance is proportional to the square root of time (t1/2) for the unmodified thread. In the presence of polysiloxanes, an increased t1/2 was observed that could be explained by the reduced flow
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rate within the cotton thread in a polysiloxanes concentration-dependent manner (Figure 2C, 2D).
3.4. Integrating polysiloxanes into thread-based microfluidic device to enhance analytical
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sensitivity. To confirm the interaction between polysiloxanes and cotton thread, their structure was
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characterized using SEM (Figure 3A). SEM revealed the interaction between thread and polysiloxanes
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that produced smaller-sized pores for the fluidic control. To evaluate the effect of different concentrations of polysiloxanes on detection sensitivity, polysiloxanes with the concentration range of 8-fold to 16-fold were tested. Along with the increased concentration of polysiloxanes, the sensitivity
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of the immunoassay improved correspondingly, as evidenced by the significantly higher optical
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density of the test zone and lower detection limit (P < 0.05) based upon the red density analysis of the test zone on the thread. The detection limits achieved by the threads supplemented with different
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concentrations of polysiloxanes are summarized in Supplementary Table 1. Including 12-fold diluted polysiloxanes into the thread achieved a detection limit down to 500 CFU/mL of S. Enteritidis, a ~10fold signal enhancement than the unmodified thread (a detection limit of ~5000 CFU/mL) (Figure 3BC). Notably, the thread supplemented with 8- and 10-fold dilution of polysiloxanes showed a higher detection limit, possibly due to the smaller pore size and porosity of the thread saturated by
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polysiloxanes. This ultimately resulted in the inability of antigen-Ab-AuNP to completely wick through the thread and react with cAb at the test zone, as evidenced by the presence of significantly higher background signal than that of the unmodified thread and that with the supplementation of a lower concentration of polysiloxanes (≥ 12-fold) (Figure 3D).
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To support the experimental data and investigate the principle of sensitivity enhancement, the phenomenon of polysiloxanes-induced reduction of fluid velocity was evaluated using mathematical
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simulation. The average velocities of the test zone were calculated, which were 0.0703, 0.014, 0.021, 0.025, 0.028 and 0.031 cm/s for unmodified thread and threads incorporated with 8-fold, 10-fold, 12fold, 14-fold and 16-fold dilutions of polysiloxanes, respectively (Figure 3E). The white arrows in the
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thread indicate the fluid movement throughout the polysiloxanes-coated region. Taken together, the
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simulation result was in agreement with the experimental data, validating that a higher concentration
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of polysiloxanes resulted in a higher flow resistance to reduce fluid velocity and affect the detection
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sensitivity of the thread-based immunoassay (Figure 3F).
Compared to the existing methods for signal enhancement in the microfluidic assays, such as enzymebased and probe-based signal enhancement [21, 24], and sample concentration [25], fluidic control
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methods are extremely simple in terms of both fabrication step and operation step (Table 2). Several
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fluidic control approaches were reported in some paper-based microfluidic studies that involved the alteration of device geometry [26] and created a hydrophobic barrier [27]. However, these approaches
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either consume a large amount of samples and/or reagents or require external equipment that reduces the unique benefits of POC application. Compared to the existing fluidic control-signal enhancement strategies, our proposed strategy allows much simpler fabrication and operation process with minimal consumption of both sample and reagent.
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3.5. Polysiloxanes-modified thread-based microfluidic device for food safety analysis. As there is an escalating need of a sensitive microfluidic device for food safety surveillance in both developed and developing countries, we further tested the ability of this thread-based microfluidic device for the detection of S. Enteritidis in food samples (i.e., whole milk, orange juice and lettuce). The threadbased immunoassay achieved a higher detection limit in spiked milk and juice (~1000 CFU/mL)
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compared to that of PBS (~500 CFU/mL), possibly due to the presence of inhibitors (e.g., calcium ions, protease, and other additives) that affect the efficiency of antibody-antigen binding (Figure 4A,
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B). For the detection of spiked lettuce, the detection limit was much higher (~5000 CFU/mL) than other food samples (Figure 4C). In this case, sample preparation steps (e.g., filtration) might remove part of the lettuce leaves prior to detection, leading to the loss of a significant amount of bacteria. In
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general, our developed device showed comparable or even more sensitive detection of Salmonella than
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the existing POC immunoassays reported in the previous literatures (e.g., ~1×105 - 1×106 CFU/mL)
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[28, 29]. The recovery levels of the proposed device are summarized in Table 1. The thread-based
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device had a high recovery level for the detection of S. Enteritidis especially in spiked whole milk and
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orange juice. To evaluate the specificity of this thread-based microfluidic device, it was tested against different species of bacteria, including E. coli O157:H7, L. monocytogenes and C. jejuni, as well as distilled water and PBS. This device showed a good specificity without a false positive result, as
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indicated by the only positive result shown in Salmonella sample (red signal at the test zone). In
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contrast, other bacterial species as well as distilled water and PBS showed negative results (no red
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signal at the test zone) (Figure 4D).
Considering the ability of polysiloxanes to resist to breaching by these surfactants with tunable hydrophobicity, we propose that this material offers a great potential to be included into the microfluidic platform for effective manipulation of fluid flow for signal enhancement. This was the first study to demonstrate the integration of polysiloxanes into the microfluidic device (i.e., thread-
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based devices) for fluidic control. The proposed device showed a number of advantages: (a) more cost effective (~$1 USD) than the conventional diagnostic platforms (e.g., spectrophotometer, microplate reader, etc.) (~$10,000 USD), (b) requires a less amount of sample and reagent (~20 μL) than the conventional test strip (>50 μL), (c) ease of operation and detection compared to the bench-top instrument, and (d) being more rapid (~10 min) than the conventional method (~1 hr) for on-site
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detection of the targeted analyte(s). Although the proposed thread-based device only allows the detection of single analyte, future work can include the detection of multiple analytes by creating
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multiple test zones along with target-specific capturing antibodies on a single thread. The device shows great promise for rapid POC applications in biomedical diagnosis, food safety surveillance, and
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environmental monitoring in a resource-limited setting.
4. Conclusions
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In summary, we proposed a new strategy of incorporating polysiloxanes with tunable hydrophobicity
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into the thread-based microfluidic devices to manipulate fluid flow for sensitivity enhancement.
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Integrating polysiloxanes into the thread-based device reduces the pore size and porosity of the entire devices, hence reducing the flow rate of biomolecules across the thread that is dependent upon the hydrophobicity of polysiloxanes. The controlled flow increases the interaction rate between the
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targeted antigen and dAb-AuNP, leading to the production of more antigen-dAb-AuNP complexes.
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These complexes eventually bind to the cAb at the test zone and produce a high color intensity at the test zone, thus improving the analytical sensitivity of the immunoassay (~10-fold signal enhancement
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than the unmodified thread-based device). Mathematical simulation validated fluid delay and was in agreement with the experimental results for different concentrations of polysiloxanes. The proposed device was able to selectively and sensitively detect S. Enteritidis in PBS, spiked milk, juice and lettuce, achieving the detection limit of 500-5000 CFU/mL. The entire sample-to-answer process needs only 10 min instead of several days required for the conventional bacterial plating assays.
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Polysiloxanes is suggested to be included into other types of microfluidic platforms (e.g., paper, cloth, etc.) to manipulate fluid flow for a variety of applications. This developed polysiloxanes-modified device has the potential to detect various targeted analytes in a sensitive and selective manner.
Acknowledgement
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This work was financially supported by funds awarded to X.L. by Natural Sciences and Engineering
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Research Council of Canada (NSERC CRDPJ 486586-15).
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Dr. Jane Ru Choi and Dr. Azadeh Nilghaz are postdoctoral fellows in Food, Nutrition and Health Program at University of British Columbia. Dr. Lei Chen is postdoctoral fellow in Department of Chemistry at University of British Columbia. Dr. Keng Chou is Associate Professor in Department of Chemistry at University of British Columbia. Corresponding author Dr. Xiaonan Lu is Associate Professor in Food, Nutrition and Health Program at University of British Columbia. He is also the director of UBC Food Safety Engineering Center.
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TABLES Table 1. Recovery levels of thread-based device for the detection of S. Enteritidis in spiked samples Detected concentration (CFU/mL)
Recovery (%)
Whole milk
5000 7500 10000
4589 ± 364.17 7200 ± 385.09 9422 ± 200.39
91.78 ± 7.28 95.99 ± 5.14 94.22 ± 2.01
Orange juice
5000 7500 10000
4311 ± 364.17 7033 ± 577.35 8978 ± 1060.03
86.22 ± 7.28 93.77 ± 7.69 89.78 ± 10.6
Lettuce
5000 7500 10000
3533 ± 254.77 5589 ± 529.87 7311 ± 771.89
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70.67 ± 5.1 74.51 ± 7.06 73.11 ± 7.72
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level
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Added concentration (CFU/mL)
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Spiked samples
Table 2. Comparison of various strategies for signal enhancement in lateral flow immunoassay
0.7 ng/mL
Complex fabrication [26] method using different sizes of pads
6.5 ng/mL
Complex device [27] fabrication using wax printing technology
10 ng/mL
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Carcinoembryonic 2.32 ng/mL antigen
Salmonella Enteritidis
100 CFU/mL
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Require extra buffer [25] addition step
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0.1 ng/µL
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Human ferritin
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Thread-based device using gold nanoparticle trimer reporter probe Thread-based device using gold nanoparticles and carbon nanotubes Thread-based device using fluidic control strategy by incorporating diluted polysiloxanes
Limit of Characteristics References detection 1.6 ng/mL Complex operation [24] steps (i.e., enzyme loading and washing steps)
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Targeted proteins Lateral flow test Human strip using gold immunoglobulin nanoparticles G (IgG) loaded with enzymes Lateral flow test Transferrin strip using sample concentration method Lateral flow test Human IgG strip using paper architecture modificationfluidic control strategy Lateral flow test Human IgG strip using wax patterned-fluidic control strategy
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Methods
Require additional [21] synthesis of nanomaterial and special probe design Require additional [30] synthesis of nanomaterial
Simple device This work fabrication and operation, lower sample and reagent consumption compared to lateral flow test strip
FIGURES Figure 1. Schematic illustration of fluidic control in the polysiloxanes-modified thread-based microfluidic device. (A) Incorporating polysiloxanes with tunable hydrophobicity into the thread enables well-controlled fluid flow that can enhance the interaction rate between biomolecules and
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subsequently improve the detection sensitivity. (B) An image of the device (scale bar=5mm).
Figure 2. The effect of polysiloxanes on fluid flow in the thread-based microfluidic device. (A)
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Eight-fold dilution of polysiloxanes and greater allow the solution to completely wick through the thread. (B) The higher the dilution of polysiloxanes, the smaller the water contact angle, indicating the less hydrophobicity of polysiloxanes-coated thread. (C-D) The higher the dilution of polysiloxanes,
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the higher the fluid flow rates and the shorter the fluidic delay period in the cotton thread.
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Figure 3. The effect of polysiloxanes on thread-based immunoassay. (A) A representative SEM
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image of unmodified (left), polysiloxanes-partially modified (middle) and polysiloxanes-modified
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(right) thread, showing the interaction between the thread and polysiloxanes (scale bar = 40 µm). (BC) Incorporating 12-fold diluted polysiloxanes into thread enables sensitive detection of bacteria (~10fold signal enhancement), achieving a detection limit of as low as 500 CFU/mL. (D) Higher
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concentration of polysiloxanes (i.e., 8- and 10-fold) shows a significantly higher background signal as
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compared to lower concentration of polysiloxanes (≥ 12-fold dilution). (E) The average velocity at the polysiloxanes-coated region. (F) Simulation of flow velocity on the thread (black arrows: limit of
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detection).
Figure 4. Testing polysiloxanes modified thread-based microfluidic device with Salmonellaspiked food and drinks. The device could sensitively detect Salmonella enterica serotype Enteritidis in (A) whole milk, (B) orange juice and (C) lettuce with the detection limit of 1000, 1000 and 5000
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CFU/mL, respectively, indicating its potential for point-of-care detection and surveillance of food safety. (D) Good specificity of the proposed device was confirmed by only positive result shown in Salmonella-positive sample while negative results were shown for other bacterial species (E. coli O157:H7, C. jejuni, and L. monocytogenes) as well as controls (distilled water and phosphate buffered
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saline) (black arrows: limit of detection).
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S0925-4005(18)30103-5 https://doi.org/10.1016/j.snb.2018.01.102 SNB 23951
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
7-9-2017 17-12-2017 9-1-2018
Please cite this article as: Jane Ru Choi, Azadeh Nilghaz, Lei Chen, Keng C.Chou, Xiaonan Lu, Modification of thread-based microfluidic device with polysiloxanes for the development of a sensitive and selective immunoassay, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.01.102 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Modification of thread-based microfluidic device with polysiloxanes for the development of a sensitive and selective immunoassay
Jane Ru Choi 1, Azadeh Nilghaz 1, Lei Chen 2, Keng C. Chou 2, and Xiaonan Lu 1*
Food, Nutrition and Health Program, Faculty of Land and Food Systems, The University of British
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Columbia, Vancouver, BC, V6T 1Z4, Canada
Department of Chemistry, The University of British Columbia, Vancouver, BC, V6T 1Z1, Canada
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Corresponding author. Email address: [email protected]
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Research highlights
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Graphical abstract
Polysiloxanes can be tuned to manipulate the fluid flow in thread-based microfluidic devices
Polysiloxanes-modified device enables optimum interaction between antigen and antibody
This device can sensitively detect bacteria in diverse real samples
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This work expands the application of polysiloxanes in microfluidics
ABSTRACT
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We demonstrate that polysiloxanes can be tuned to be partially hydrophilic for manipulating the fluidic flow in the pores of cotton thread-based microfluidic devices. A mixture of methanol and isopropanol was used as a diluent for siloxane precursor, which was included into the thread to enable rapid curing of polysiloxanes for fluidic control and enhance detection sensitivity. Interestingly, twelve-fold diluted polysiloxanes enabled desirable fluidic delay and optimum interaction between the targeted antigen
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and detection antibody-gold nanoparticles (dAb-AuNPs) in the thread-based immunoassay, generating more antigen-dAb-AuNP complexes that bound to the capture antibody (cAb) at the test zone and
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achieved signal enhancement (~10-fold over unmodified device). The phenomenon of fluidic delay was evaluated by mathematical simulation, through which the fluidic movement on the polysiloxanescoated region was observed and the simulation data was in agreement with the experimental data. This
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polysiloxanes-modified device could detect Salmonella enterica serotype Enteritidis (as a model
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analyte) in phosphate buffered saline, spiked whole milk, juice and lettuce with the detection limit of
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500, 1000, 1000 and 5000 CFU/mL, respectively, which was comparable to or even more sensitive
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than the existing immunoassays. This work expands the application of polysiloxanes in the
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microfluidic devices for biomedical diagnosis, water quality monitoring, and food safety surveillance.
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immunoassay
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Keywords: polysiloxanes, thread-based microfluidic devices, fluidic control, sensitivity enhancement,
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1. Introduction With the advances in point-of-care (POC) technologies, paper-based and textile-based microfluidic devices have become attractive platforms for nucleic acid testing and immunoassays for a wide range of applications (e.g., biomedical diagnosis, food safety surveillance, and environmental monitoring) [1]. In general, these POC assays are complementary to the conventional labor-intensive, time-
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consuming and expensive laboratory diagnostic assays, such as enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) technique [2, 3]. Textile-based
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devices (e.g., thread-based devices) are of extremely lower-cost (<$1 USD per test) and require a lower sample volume (~20 μL) for testing compared to lateral flow test strip (require ~50 μL sample, $3 USD per test) [4]. The nitrocellulose membrane of lateral flow test strip made up of cellulose nitrate
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is more fragile and inherently brittle under tension compared to thread. Similar to paper substrates, the
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cotton thread has excellent capabilities of immobilizing biomolecules (e.g., proteins and nucleic acids)
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and transporting fluid via capillary action, hence it is highly suitable for rapid detection and analysis
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of various analytes [5]. The excellent properties of thread make it a promising alternative to the
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conventional lateral flow test strips [5-7]. However, similar to paper-based microfluidic devices, the relatively poor sensitivity of the thread-based microfluidic devices has limited their wide applications
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[8].
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Polysiloxanes are inorganic polymers that consist of repeating units of siloxane and also known as silicones. They have the ability to resist to breaching by liquids and surfactants with low surface energy
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[9]. Their compatibility to various chemicals makes them a possible candidate to be included into the microfluidic devices for the development of various chemical and biological assays. Other hydrophobic materials, such as wax and alkyl ketene dimer, are commonly used in the development of different microfluidic assays [10]. However, they are unable to resist to breaching by the low surface energy surfactants [9]. The examples of surfactants include anionic (e.g., sodium dodecyl sulfate),
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non-ionic (e.g., Triton X-100) and cationic (e.g., cetyltrimethylammonium bromide) surfactants, which are commonly used as lysis reagents in most biological assays [11, 12]. In contrast, polysiloxanes has been reported to have the ability to resist to breaching by these surfactants due to its relatively lower surface energy [9]. For the synthesis of polysiloxanes, the Piers-Rubinsztajn reaction utilizes a Lewis acidic boron catalyst to facilitate the condensation of hydrosilanes with alkoxysilanes
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that can form new siloxane bonds [13]. This reaction eventually leads to the production of polysiloxanes only in a few seconds or minutes at room temperature [9]. Despite its ability to create
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hydrophobic zones and channels in the microfluidic assays due to the aforementioned properties, the possibility of tuning its hydrophobicity in the microfluidic assays has remained unexplored yet. Considering the potential of tuning its hydrophobicity, incorporating it into the thread-based device
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can potentially achieve desirable fluidic control and optimum device performance at a minimal cost.
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In the current study, we demonstrated a new approach of tuning the hydrophobicity of polysiloxanes
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to manipulate liquid flow in a thread-based microfluidic assay. A mixture of methanol and isopropanol
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was used as a diluent for siloxane precursor, which was then incorporated into the cotton thread-based devices to enable rapid curing of polysiloxanes for fluidic control within their pores. Interestingly, we identified that the delay of fluid flow was dependent upon the concentration of polysiloxanes, and the
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controlled flow increased the interaction between the targeted antigens and detection antibodies-gold
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nanoparticles (dAb-AuNPs) in thread-based assays, forming more antigen-dAb-AuNP complexes. These complexes eventually bound to the capture Ab (cAb) at the test zone and enabled ~10-fold signal
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enhancement over the unmodified thread-based immunoassay. The phenomenon of fluidic delay was evaluated by mathematical simulation through which the fluidic movement on the polysiloxanescoated region was observed and the simulation result was in agreement with the experimental data. As food microbiological safety remains a major concern of public health in both developing and developed countries, the application of our developed device for food safety analysis was evaluated
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by detecting foodborne pathogens in spiked drink and food samples. Salmonella enterica serotype Enteritidis was used as a model bacterium in this study given that it is one of the most common causes of foodborne illnesses worldwide [14]. S. Enteritidis could be detected by using this polysiloxanesmodified thread-based device in phosphate buffered saline, spiked whole milk, juice and lettuce with the detection limit of 500, 1000, 1000 and 5000 CFU/mL, respectively, which was comparable to or
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even more sensitive than the current POC immunoassays. The good specificity of this proposed immunoassay was evidenced by only positive result shown in Salmonella-positive samples. In
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comparison, other bacterial samples (i.e., Escherichia coli, Campylobacter, and Listeria monocytogenes) and controls [i.e., distilled water and phosphate buffered saline (PBS)] showed
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negative results.
2. Materials and Methods
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2.1. Bacterial strains and cultivation. S. Enteritidis ATCC 43353, E. coli O157:H7 ATCC 43894, C.
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jejuni F38011 (a clinical isolate) and L. monocytogenes ATCC 19113 were used in the current study.
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S. Enteritidis and E. coli O157:H7 were cultivated in tryptic soy broth (BD Difco) while L. monocytogenes was cultivated in Brain Heart Infusion (BHI) broth (BD Difco) in an aerobic condition. C. jejuni was cultivated in Mueller Hinton broth (BD Difco) in a microaerobic condition (85% N2,
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10% CO2, 5% O2). The cultivation temperature of all the bacteria was set at 37°C.
2.2. Preparation of gold nanoparticles and gold nanoparticles-antibody conjugates. Gold
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nanoparticles (AuNPs) with an average diameter of 13±3 nm were prepared according to the protocol described in previous publications [1, 2]. Briefly, 4.5 mL of 1% trisodium citrate solution and 1% chloroauric acid (HAuCl4) was rapidly added into a round bottom flask with 100 mL of boiled distilled water. The color of the solution immediately turned from yellow to purple and finally to wine red in a few minutes.
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2.3. Preparation of gold nanoparticles-antibody conjugates. The optimum pH value of 8 was achieved by adding 4 µL of potassium carbonate into the AuNP solution. Different volumes of dAb (anti-S. Enteritidis mouse monoclonal Ab, 0, 1, 2, 3, 4, and 5 µL) (VWR Scientific Canada) were added into the AuNP solution in different tubes. Then, 10% (w/v) BSA was added to block non-
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specific binding sites. The supernatant was removed after the centrifugation at 12,000 ×g for 10 min, and the red pellets were suspended in 0.22 mL of eluent buffer that consists of 1% BSA, 0.85% Tris,
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5% trehalose, and 20% sucrose. The AuNPs were characterized using transmission electron microscopy (TEM) whereas the AuNP-antibody conjugates were characterized by the visible colour changes from wine red to dark red and a slight shift of the absorbance values in ultraviolet-visible
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spectrophotometry.
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2.4. Fabrication of the thread-based microfluidic device. The thread-based microfluidic device was
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fabricated according to the protocols described in a previous publication with modifications [3].
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Briefly, the thread-based device consisted of three major components: a sample pad, a cotton thread and an absorbent pad. Cotton threads were treated to be hydrophilic with air plasma (PDC-32G, Harrick Plasma, Ithaca, NY). A test zone and a control zone were defined on each thread. The test
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zone was coated with 0.6 µL (applied as three aliquots of 0.2 µL, with 10 min drying at 37°C after the
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addition of each aliquot) of cAb against S. Enteritidis at a concentration of 1 mg/mL in PBS containing 15% glycerol. The control zone was coated with anti-mouse IgG (0.6 µL at 1 mg/mL) (VWR Scientific
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Canada). The antibody was applied as three aliquots of 0.2 µL, with 10 min drying at 37°C after the addition of each aliquot. Five microliters of AuNP-Ab were added onto the sample pad and dried at 37°C for 1 hr before use. Double-sided tapes were placed on the backing pad in parallel ways with a distance up to 4 cm. The thread was then mounted onto the double-sided tapes. A piece of glass fiber
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that served as sample pad (10 × 10 mm) was placed at one end of the thread whereas a piece of cellulose pad (10 × 20 mm) that served as absorbent pad was placed at the other end.
2.5. Preparation of the polysiloxane-modified thread-based microfluidic device. Polysiloxane was used to control the fluidic flow of the immunoassay. This hydrophobic agent was prepared based upon
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the protocol described in a previous publication with modifications [3]. Briefly, 0.004 mmol tris(pentafluorophenyl)borane [B(C6F5)3] was added to 1,3-dimethyltetramethoxysilane (DMTMDS)
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(0.6 mmol) to form the stock solution. The stock solution was mixed with the appropriate amount of methanol-isopropanol mixture and finally with tetrakis(dimethylsiloxy)silane (QMH4, Gelest). Different concentrations of hydrophobic mixture (0.5 µL) were then individually dispensed into the
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threads.
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2.6. Optimization of the thread-based immunoassay. In the optimization assays, the structure of
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thread-based device, the volume of dAb-AuNP, as well as the concentration of glycerol and cAb at the
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test zone were optimized to achieve the optimum colorimetric signal at the test zone. Different constructions of the thread-based device were performed, especially on both test zone and control zone in order to minimize the spreading area of both cAb and control Ab and simplify assay readout. To
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optimize the concentration of dAb-AuNP, different volumes of dAb-AuNP (1, 2, 3, 4, and 5 µL) were
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individually tested and the optimum volume of dAb-AuNP was determined. To optimize the concentration of glycerol at the test zone, different concentrations of glycerol (5%, 10%, 15%, and
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20%) were individually mixed with cAb and the corresponding color intensity was observed. Different concentrations of cAb at the test zone (0.5, 1, 1.5, and 2 mg/mL) were also individually tested in the immunoassay. The sensitivity of the assay was determined based upon the color intensity at the test zone and the optimum parameters were determined.
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2.7. Thread-based lateral flow immunoassay for the detection of S. Enteritidis. To perform the lateral flow immunoassay, 20 µL of sample was applied onto the sample pad. Sensitivity assay was performed with different concentrations of polysiloxanes using a mixture of methanol and isopropanol as the diluent. The specificity of the assay was determined by testing the device using different species of bacteria (i.e., E. coli O157:H7, L. monocytogenes and C. jejuni). The color changes at both test zone
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and control zone were observed in 10 min. For quantitative analysis, images of the test zones were
ProPlus 6.0 software (Media cybernetics. Inc., Bethesda, MD).
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captured using a smartphone (IPhone 6S) and their optical densities were determined using the image
2.8. Mathematical simulation. To mathematically simulate the fluidic delay due to polysiloxanes in
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the thread, a physical model for a steady-state flow was developed in the current study. Brinkman
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equation was used to describe the fluidic flow in this porous material and the viscosity effect is taken
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into consideration:
, where
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(1)
, p, µ, µe and K are fluid velocity, pressure, fluid viscosity, effective viscosity for Brinkman
term and permeability of porous medium, respectively. The fluid viscosity (µ) corresponds to that of
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PBS (as a diluent) at room temperature. The effective viscosity (µe) corresponds to the fluid viscosity
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(µ) in the porous medium [15]. The permeability K is given as follows [16]:
K =r 2
1 1
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1.5
2
(2)
, where ε is the porosity of the material and r is the average fiber radius. The porosity ε was obtained by measuring the volume of the liquid absorbed by the materials based upon the liquid-displacement method with modifications [17]. The average fiber radius r for each group were obtained from the 9
scanning electron microscopy (SEM) images, which were 17±0.12 µm, 13.3±0.15 µm, 12±0.26 µm, 11.23±0.15 µm, 10.4±0.12 µm and 8.2±0.34 µm for unmodified thread and the threads incorporated with 8-fold, 10-fold, 12-fold, 14-fold and 16-fold dilutions of polysiloxanes, respectively. As for the boundary conditions, the inlet velocity was calculated with the known sample volume (20 µL), the inlet cross-section and the fluid absorption period. The outlet pressure is equivalent to the atmospheric
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pressure. All other bounding walls are under non-slip conditions. The mathematical simulation was
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performed using the Brinkman equation module of Comsol Multiphysics 5.0 software.
2.9. Detection of S. Enteritidis in different types of food samples. After overnight cultivation, bacteria were first diluted in PBS for optimization of the thread-based assays. To further investigate
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the ability of the device to detect pathogens in agri-food products, we spiked S. Enteritidis into milk,
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juice and lettuce obtained from a local grocery store in Vancouver, Canada. Each sample was first
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confirmed to be without contamination of Salmonella, followed by spiking different concentrations of
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S. Enteritidis (i.e., ranging from 1 to 105 CFU/mL). Lettuces were washed and then spiked with S.
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Enteritidis before mixing with 100 mL of ultrapure water in a blender. The mixture was then filtered through a 100-µm filter paper to remove the debris prior to the detection by the immunoassay.
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3. Results and Discussion
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3.1. Working principle of polysiloxanes-modified thread-based microfluidic device. Taking the advantage of polysiloxanes that resist to the surfactants, we propose that its hydrophobicity can be
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tuned and its incorporation into the thread-based microfluidic device can manipulate fluid flow and subsequently achieve the optimum performance of the device. The Piers-Rubinsztajn reaction was carried out for the formation of silicone elastomer to prepare polysiloxanes [13]. This reaction is rapid and utilizes a Lewis acidic boron catalyst namely tris(pentafluorophenyl)borane, B(C6F5)3 to facilitate the condensation of hydrosilanes [tetrakis(dimethylsiloxy)silane] with alkoxysilanes (1,3-
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dimethyltetramethoxysilane) to form new siloxane bonds (R3SiH + R3SiOR’ → R3SiOSiR3 + R’H) [9, 18]. As aforementioned, a mixture of methanol and isopropanol (1:1) was used as a diluent for siloxane precursor as it can ideally control both viscosity and surface tension of the final products, namely polysiloxanes. By tuning the amount of diluent, we propose that the hydrophobicity of polysiloxanes can be tuned to enable a well-controlled fluidic flow in the thread-based microfluidic
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device.
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Similar to paper-based immunoassays, thread-based immunoassays consist of two major zones, namely test zone and control zone. The cAb and control Ab were dispensed onto the test zone and control zone, respectively. Cotton thread used in the study is mainly made up of cellulose. There are
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high density of hydroxyl groups on the surface of cellulose that allow the irreversible binding of
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capturing antibody to the surface of cotton thread through a combination of electrostatic and
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hydrophobic interactions [19, 20]. These interactions are sufficiently strong to resist to the fluidic
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movement. The AuNPs used in this study were characterized using transmission electron microscopy
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(TEM) (Figure S1A). The AuNP-antibody conjugates were characterized by the visible colour changes from wine red to dark red (Figure S1B) and a slight shift of the absorbance values (7 nm) in
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ultraviolet-visible spectrophotometry (Figure S1C). The particle size was 13 nm (Figure S1D).
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The cotton thread is attached to the sample pad that allows sample addition and AuNP release as well as absorbent pad that facilitates fluid flow. Polysiloxanes was added onto the region between sample
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pad and test zone to create fluidic delay. Twenty microliters of sample (i.e., Salmonella) were applied onto the sample pad where dAb-AuNP was dried. The antigen-dAb-AuNP complexes then flowed across the cotton thread and bound to the cAb on the test zone to form colorimetric band. The color intensity of the test zone was directly proportional to the concentration of the analytes (i.e., Salmonella). The excessive dAb-AuNP was bound to the control zone and formed another colorimetric
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band (Figure 1).
3.2. Optimization of the thread-based immunoassay. To reduce the spreading area of cAb and control Ab at both zones, the regions of the test zone and control zone were modified. Both zones should be small and concentrated to minimize antibody spreading in the immunoassay. Unlike
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previous publications that demonstrated the direct dispension of antibodies onto the thread [19, 21, 22], we modified both zones by creating knots to reduce the spreading area of antibodies (Figure S2).
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To create a knot, one end of the thread was inserted into the loop and both ends were pulled together in the opposite direction to tighten it. The other knot was also created using the same method. This modified structure simplifies the readout and assay analysis by clearly defining the region of the
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colorimetric signal. We also dispensed three aliquots (0.2 µL each) to further minimize the wicking of
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the antibody solution along the thread. To determine the amount of dAb-AuNP that was required to
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achieve the optimum colorimetric signal at the test zone, different volumes of dAb-AuNP (i.e., 1-5
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µL) were individually tested (Figure S3). The amount that required obtaining an optimum signal in
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the immunoassay was determined to be 4 µL. A smaller volume of the dAb-AuNP produced a weaker signal at the test zone while a larger volume yielded an increase in the background signal.
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To determine the concentration of the glycerol that acts as a diluent of dAb-AuNP, we optimized its
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concentration in the range of 5% - 20%. In general, 15% of glycerol produced an optimum signal at the test zone. Glycerol with a lower concentration (<15%) yielded a weaker signal while glycerol with
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a higher concentration (i.e., 20%) resulted in an increase in the background signal. This might be due to the significantly increased viscosity at the test zone by the treatment of a high concentration of glycerol, leading to the failure of considerable amount of antigen-dAb-AuNP complexes to completely wick through the thread that subsequently generated a false positive result (Figure S3).
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The concentration of the immobilized cAb at the test zone was also tested in the range of 0.5-2 mg/mL (Figure S3). Along with the increased concentration of cAb, the color intensity of the test zone increased. However, a concentration over 1.5 mg/mL (i.e., 2 mg/mL) produced a false positive result with a higher background signal, which might be due to the significant amount of cAb that reduced the flow rate of antigen-dAb-AuNP complexes. Therefore, 1.5 mg/mL was selected as the optimum
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concentration of cAb. The optimized conditions (i.e., 4 μL dAb-AuNP, 15% glycerol and 1.5 mg/mL immobilized cAb) with the modified “knot” structure of the thread were then used for the subsequent
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assays, which were able to produce intensive color at the test zone with negligible background signal.
3.3. Integrating polysiloxanes into thread-based microfluidic device to create fluidic delay. We
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utilized the optimum volume of polysiloxanes (0.5 μL) to completely cover the biomolecule interaction
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region (i.e., the entire region prior to the test zone) for desirable manipulation of the flow rate (Figure
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S4). To evaluate the effect of polysiloxanes on thread-based immunoassay, polysiloxanes were
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dispensed into the thread prior to running the assay. To determine the range of the concentration
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required to allow the fluid to wick through the entire thread, we performed the immunoassay with polysiloxanes in the concentration range of 4-fold to 16-fold dilution (Figure 2A). The concentration of polysiloxanes with at least 8-fold dilution allowed the solution to completely flow through the
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thread. The 4-fold diluted polysiloxanes was too concentrated to allow the complete flow of liquid
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within the thread. Therefore, we utilized polysiloxanes with the concentration range of 8-fold to 16-
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fold for the development of this immunoassay.
To study the effect of different concentrations of polysiloxanes on the hydrophobicity of the thread, contact angle measurements were performed. In the absence of polysiloxanes (negative control), water droplets were identified to immediately wick into the thread. In contrast, the highly concentrated polysiloxanes allowed the thread to be more hydrophobic, thereby a water droplet being formed on the
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surface of the thread with an apparent contact angle. The contact angle of droplets was 89.6 ± 6°, 65.7 ± 2°, 56.3 ± 1°, 52.2 ± 6° 46.5 ± 4° and 34.9 ± 8° for 4-fold, 8-fold, 10-fold, 12-fold, 14-fold and 16fold dilution of polysiloxanes, respectively, at 2 s after the addition of 5 μL of liquids (Figure 2B). Taken together, the lower the concentration of polysiloxanes on the thread, the lower the water contact angle, indicating less hydrophobicity of the polysiloxanes-coated thread. To study the hydrophobicity
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of polysiloxanes on the flow rate, different dilutions of polysiloxanes were applied onto the thread and their flow distance over time was correspondingly recorded. Based upon the Washburn’s equation
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[23], the flow distance is proportional to the square root of time (t1/2) for the unmodified thread. In the presence of polysiloxanes, an increased t1/2 was observed that could be explained by the reduced flow
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rate within the cotton thread in a polysiloxanes concentration-dependent manner (Figure 2C, 2D).
3.4. Integrating polysiloxanes into thread-based microfluidic device to enhance analytical
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sensitivity. To confirm the interaction between polysiloxanes and cotton thread, their structure was
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characterized using SEM (Figure 3A). SEM revealed the interaction between thread and polysiloxanes
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that produced smaller-sized pores for the fluidic control. To evaluate the effect of different concentrations of polysiloxanes on detection sensitivity, polysiloxanes with the concentration range of 8-fold to 16-fold were tested. Along with the increased concentration of polysiloxanes, the sensitivity
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of the immunoassay improved correspondingly, as evidenced by the significantly higher optical
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density of the test zone and lower detection limit (P < 0.05) based upon the red density analysis of the test zone on the thread. The detection limits achieved by the threads supplemented with different
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concentrations of polysiloxanes are summarized in Supplementary Table 1. Including 12-fold diluted polysiloxanes into the thread achieved a detection limit down to 500 CFU/mL of S. Enteritidis, a ~10fold signal enhancement than the unmodified thread (a detection limit of ~5000 CFU/mL) (Figure 3BC). Notably, the thread supplemented with 8- and 10-fold dilution of polysiloxanes showed a higher detection limit, possibly due to the smaller pore size and porosity of the thread saturated by
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polysiloxanes. This ultimately resulted in the inability of antigen-Ab-AuNP to completely wick through the thread and react with cAb at the test zone, as evidenced by the presence of significantly higher background signal than that of the unmodified thread and that with the supplementation of a lower concentration of polysiloxanes (≥ 12-fold) (Figure 3D).
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To support the experimental data and investigate the principle of sensitivity enhancement, the phenomenon of polysiloxanes-induced reduction of fluid velocity was evaluated using mathematical
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simulation. The average velocities of the test zone were calculated, which were 0.0703, 0.014, 0.021, 0.025, 0.028 and 0.031 cm/s for unmodified thread and threads incorporated with 8-fold, 10-fold, 12fold, 14-fold and 16-fold dilutions of polysiloxanes, respectively (Figure 3E). The white arrows in the
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thread indicate the fluid movement throughout the polysiloxanes-coated region. Taken together, the
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simulation result was in agreement with the experimental data, validating that a higher concentration
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of polysiloxanes resulted in a higher flow resistance to reduce fluid velocity and affect the detection
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sensitivity of the thread-based immunoassay (Figure 3F).
Compared to the existing methods for signal enhancement in the microfluidic assays, such as enzymebased and probe-based signal enhancement [21, 24], and sample concentration [25], fluidic control
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methods are extremely simple in terms of both fabrication step and operation step (Table 2). Several
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fluidic control approaches were reported in some paper-based microfluidic studies that involved the alteration of device geometry [26] and created a hydrophobic barrier [27]. However, these approaches
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either consume a large amount of samples and/or reagents or require external equipment that reduces the unique benefits of POC application. Compared to the existing fluidic control-signal enhancement strategies, our proposed strategy allows much simpler fabrication and operation process with minimal consumption of both sample and reagent.
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3.5. Polysiloxanes-modified thread-based microfluidic device for food safety analysis. As there is an escalating need of a sensitive microfluidic device for food safety surveillance in both developed and developing countries, we further tested the ability of this thread-based microfluidic device for the detection of S. Enteritidis in food samples (i.e., whole milk, orange juice and lettuce). The threadbased immunoassay achieved a higher detection limit in spiked milk and juice (~1000 CFU/mL)
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compared to that of PBS (~500 CFU/mL), possibly due to the presence of inhibitors (e.g., calcium ions, protease, and other additives) that affect the efficiency of antibody-antigen binding (Figure 4A,
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B). For the detection of spiked lettuce, the detection limit was much higher (~5000 CFU/mL) than other food samples (Figure 4C). In this case, sample preparation steps (e.g., filtration) might remove part of the lettuce leaves prior to detection, leading to the loss of a significant amount of bacteria. In
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general, our developed device showed comparable or even more sensitive detection of Salmonella than
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the existing POC immunoassays reported in the previous literatures (e.g., ~1×105 - 1×106 CFU/mL)
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[28, 29]. The recovery levels of the proposed device are summarized in Table 1. The thread-based
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device had a high recovery level for the detection of S. Enteritidis especially in spiked whole milk and
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orange juice. To evaluate the specificity of this thread-based microfluidic device, it was tested against different species of bacteria, including E. coli O157:H7, L. monocytogenes and C. jejuni, as well as distilled water and PBS. This device showed a good specificity without a false positive result, as
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indicated by the only positive result shown in Salmonella sample (red signal at the test zone). In
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contrast, other bacterial species as well as distilled water and PBS showed negative results (no red
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signal at the test zone) (Figure 4D).
Considering the ability of polysiloxanes to resist to breaching by these surfactants with tunable hydrophobicity, we propose that this material offers a great potential to be included into the microfluidic platform for effective manipulation of fluid flow for signal enhancement. This was the first study to demonstrate the integration of polysiloxanes into the microfluidic device (i.e., thread-
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based devices) for fluidic control. The proposed device showed a number of advantages: (a) more cost effective (~$1 USD) than the conventional diagnostic platforms (e.g., spectrophotometer, microplate reader, etc.) (~$10,000 USD), (b) requires a less amount of sample and reagent (~20 μL) than the conventional test strip (>50 μL), (c) ease of operation and detection compared to the bench-top instrument, and (d) being more rapid (~10 min) than the conventional method (~1 hr) for on-site
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detection of the targeted analyte(s). Although the proposed thread-based device only allows the detection of single analyte, future work can include the detection of multiple analytes by creating
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multiple test zones along with target-specific capturing antibodies on a single thread. The device shows great promise for rapid POC applications in biomedical diagnosis, food safety surveillance, and
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environmental monitoring in a resource-limited setting.
4. Conclusions
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In summary, we proposed a new strategy of incorporating polysiloxanes with tunable hydrophobicity
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into the thread-based microfluidic devices to manipulate fluid flow for sensitivity enhancement.
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Integrating polysiloxanes into the thread-based device reduces the pore size and porosity of the entire devices, hence reducing the flow rate of biomolecules across the thread that is dependent upon the hydrophobicity of polysiloxanes. The controlled flow increases the interaction rate between the
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targeted antigen and dAb-AuNP, leading to the production of more antigen-dAb-AuNP complexes.
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These complexes eventually bind to the cAb at the test zone and produce a high color intensity at the test zone, thus improving the analytical sensitivity of the immunoassay (~10-fold signal enhancement
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than the unmodified thread-based device). Mathematical simulation validated fluid delay and was in agreement with the experimental results for different concentrations of polysiloxanes. The proposed device was able to selectively and sensitively detect S. Enteritidis in PBS, spiked milk, juice and lettuce, achieving the detection limit of 500-5000 CFU/mL. The entire sample-to-answer process needs only 10 min instead of several days required for the conventional bacterial plating assays.
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Polysiloxanes is suggested to be included into other types of microfluidic platforms (e.g., paper, cloth, etc.) to manipulate fluid flow for a variety of applications. This developed polysiloxanes-modified device has the potential to detect various targeted analytes in a sensitive and selective manner.
Acknowledgement
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This work was financially supported by funds awarded to X.L. by Natural Sciences and Engineering
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Research Council of Canada (NSERC CRDPJ 486586-15).
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[20] G. Zhou, X. Mao, D. Juncker, Immunochromatographic assay on thread, Anal Chem, 84(2012) 7736-43. [21] X. Mao, T.-E. Du, L. Meng, T. Song, Novel gold nanoparticle trimer reporter probe combined with dry-reagent cotton thread immunoassay device for rapid human ferritin test, Anal Chim Acta, 889(2015) 172-8. [22] L.-L. Meng, T.-T. Song, X. Mao, Novel immunochromatographic assay on cotton thread based on carbon nanotubes reporter probe, Talanta, 167(2017) 379-84. [23] B.J. Toley, B. McKenzie, T. Liang, J.R. Buser, P. Yager, E. Fu, Tunable-delay shunts for paper microfluidic devices, Anal Chem, 85(2013) 11545-52. [24] C. Parolo, A. de la Escosura-Muñiz, A. Merkoçi, Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes, Biosens Bioelectron, 40(2013) 412-6. [25] R.Y. Chiu, E. Jue, A.T. Yip, A.R. Berg, S.J. Wang, A.R. Kivnick, et al., Simultaneous concentration and detection of biomarkers on paper, Lab on a chip, 14(2014) 3021-8. [26] C. Parolo, M. Medina-Sánchez, A. de la Escosura-Muñiz, A. Merkoçi, Simple paper architecture modifications lead to enhanced sensitivity in nanoparticle based lateral flow immunoassays, Lab on a chip, 13(2013) 386-90. [27] L. Rivas, M. Medina-Sanchez, A. de la Escosura-Muniz, A. Merkoci, Improving sensitivity of gold nanoparticle-based lateral flow assays by using wax-printed pillars as delay barriers of microfluidics, Lab on a chip, 14(2014) 4406-14. [28] P. Preechakasedkit, K. Pinwattana, W. Dungchai, W. Siangproh, W. Chaicumpa, P. Tongtawe, et al., Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum, Biosens Bioelectron, 31(2012) 562-6. [29] W. Wang, L. Liu, S. Song, L. Tang, H. Kuang, C. Xu, A highly sensitive ELISA and immunochromatographic strip for the detection of Salmonella typhimurium in milk samples, Sensors, 15(2015) 5281-92. [30] X. Jia, T. Song, Y. Liu, L. Meng, X. Mao, An immunochromatographic assay for carcinoembryonic antigen on cotton thread using a composite of carbon nanotubes and gold nanoparticles as reporters, Anal Chim Acta, 969(2017) 57-62.
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Dr. Jane Ru Choi and Dr. Azadeh Nilghaz are postdoctoral fellows in Food, Nutrition and Health Program at University of British Columbia. Dr. Lei Chen is postdoctoral fellow in Department of Chemistry at University of British Columbia. Dr. Keng Chou is Associate Professor in Department of Chemistry at University of British Columbia. Corresponding author Dr. Xiaonan Lu is Associate Professor in Food, Nutrition and Health Program at University of British Columbia. He is also the director of UBC Food Safety Engineering Center.
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TABLES Table 1. Recovery levels of thread-based device for the detection of S. Enteritidis in spiked samples Detected concentration (CFU/mL)
Recovery (%)
Whole milk
5000 7500 10000
4589 ± 364.17 7200 ± 385.09 9422 ± 200.39
91.78 ± 7.28 95.99 ± 5.14 94.22 ± 2.01
Orange juice
5000 7500 10000
4311 ± 364.17 7033 ± 577.35 8978 ± 1060.03
86.22 ± 7.28 93.77 ± 7.69 89.78 ± 10.6
Lettuce
5000 7500 10000
3533 ± 254.77 5589 ± 529.87 7311 ± 771.89
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70.67 ± 5.1 74.51 ± 7.06 73.11 ± 7.72
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level
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Added concentration (CFU/mL)
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Spiked samples
Table 2. Comparison of various strategies for signal enhancement in lateral flow immunoassay
0.7 ng/mL
Complex fabrication [26] method using different sizes of pads
6.5 ng/mL
Complex device [27] fabrication using wax printing technology
10 ng/mL
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Carcinoembryonic 2.32 ng/mL antigen
Salmonella Enteritidis
100 CFU/mL
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Require extra buffer [25] addition step
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0.1 ng/µL
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Human ferritin
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Thread-based device using gold nanoparticle trimer reporter probe Thread-based device using gold nanoparticles and carbon nanotubes Thread-based device using fluidic control strategy by incorporating diluted polysiloxanes
Limit of Characteristics References detection 1.6 ng/mL Complex operation [24] steps (i.e., enzyme loading and washing steps)
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Targeted proteins Lateral flow test Human strip using gold immunoglobulin nanoparticles G (IgG) loaded with enzymes Lateral flow test Transferrin strip using sample concentration method Lateral flow test Human IgG strip using paper architecture modificationfluidic control strategy Lateral flow test Human IgG strip using wax patterned-fluidic control strategy
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Methods
Require additional [21] synthesis of nanomaterial and special probe design Require additional [30] synthesis of nanomaterial
Simple device This work fabrication and operation, lower sample and reagent consumption compared to lateral flow test strip
FIGURES Figure 1. Schematic illustration of fluidic control in the polysiloxanes-modified thread-based microfluidic device. (A) Incorporating polysiloxanes with tunable hydrophobicity into the thread enables well-controlled fluid flow that can enhance the interaction rate between biomolecules and
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subsequently improve the detection sensitivity. (B) An image of the device (scale bar=5mm).
Figure 2. The effect of polysiloxanes on fluid flow in the thread-based microfluidic device. (A)
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Eight-fold dilution of polysiloxanes and greater allow the solution to completely wick through the thread. (B) The higher the dilution of polysiloxanes, the smaller the water contact angle, indicating the less hydrophobicity of polysiloxanes-coated thread. (C-D) The higher the dilution of polysiloxanes,
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the higher the fluid flow rates and the shorter the fluidic delay period in the cotton thread.
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Figure 3. The effect of polysiloxanes on thread-based immunoassay. (A) A representative SEM
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image of unmodified (left), polysiloxanes-partially modified (middle) and polysiloxanes-modified
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(right) thread, showing the interaction between the thread and polysiloxanes (scale bar = 40 µm). (BC) Incorporating 12-fold diluted polysiloxanes into thread enables sensitive detection of bacteria (~10fold signal enhancement), achieving a detection limit of as low as 500 CFU/mL. (D) Higher
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concentration of polysiloxanes (i.e., 8- and 10-fold) shows a significantly higher background signal as
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compared to lower concentration of polysiloxanes (≥ 12-fold dilution). (E) The average velocity at the polysiloxanes-coated region. (F) Simulation of flow velocity on the thread (black arrows: limit of
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detection).
Figure 4. Testing polysiloxanes modified thread-based microfluidic device with Salmonellaspiked food and drinks. The device could sensitively detect Salmonella enterica serotype Enteritidis in (A) whole milk, (B) orange juice and (C) lettuce with the detection limit of 1000, 1000 and 5000
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CFU/mL, respectively, indicating its potential for point-of-care detection and surveillance of food safety. (D) Good specificity of the proposed device was confirmed by only positive result shown in Salmonella-positive sample while negative results were shown for other bacterial species (E. coli O157:H7, C. jejuni, and L. monocytogenes) as well as controls (distilled water and phosphate buffered
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saline) (black arrows: limit of detection).
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29
EP
CC
A TE D
IP T
SC R
U
N
A
M